The present invention relates to a process and apparatus for freezing living cells.
In recent years processes for the treatment of cancers and tumors have been developed in which, after chemotherapy, the patient is transfused with specific body cells. Since it is not possible, because of the danger of fatal rejection, to use cells of donors other than the patient himself, it has been necessary to remove the specific body cell from the patient, prior to treatment and then store such cells until they are needed. To this end it is necessary to store the withdrawn cells for prolonged periods, often several months. To preserve such cells cryogenic preservation at low temperatures, have been employed since it is yet no other means available to store living cells.
Relatively large quantities of such cells are needed for the post-therapy infusion and most importantly, such large quantities must have a relatively large ratio of living cells. Nevertheless, none of the known cryogenic freezing processes is capable of freezing large cell quantities in a single sample nor are such processes capable of maintaining the high levels of living cells required for optimum therapeutic purposes. While the importance of improved cancer therapy might justify a high cost level, it is almost impossible, no matter at what cost, with the current processes to obtain the required large quantity of cells as for example autologous lymphoid blood corpuscles or medullar cells.
There are several factors in the cryogenic preservation processes which are extremely important. Among these are the steps of:
(1) The taking of the cell sample from the blood, marrow or tissue and the concentration therefrom of the required cell type;
(2) The transfer of this concentration to a freezing container;
(3) The mixing of the concentrated sample with a freeze protectant;
(4) The controlled freezing of the specific sample;
(5) Prolonged storage below minus 130.degree. C. (143.degree. K.); and
(6) Thawing of the sample in precise time/temperature relationship; and revitalization of the cells, i.e., their gradual dilution and elutriation.
Of the foregoing factors the most vital step and the one which has up until now presented the greatest difficulty is the controlled freezing of the sample.
According to the known freezing processes the cell samples were placed in closed freezing chambers wherein the temperature was reduced at a constant rate. It was found, however, that the cooling curve of the sample did not conform to the linear curve of the temperature drop in the cooling chamber, nor did the cooling curve of the temperature take into account the cooling curve of the sample, but to the curve as seen in FIG. 1. The sample would initially follow the curve of the temperature of the chamber, until a point below the freezing level T.sub.F at which time it would rise to a plateau defined at its upper limit by the freezing temperature T.sub.F at its lower limit T.sub.P (phase transformation temperature) constituting a pleateau where it would remain for some time. After some minutes, the temperature of the sample would again drop below the plateau T.sub.P at an extremely steep descent until it almost reaches the curve of the chamber temperature and thereafter follows in parallel the curve of the chamber temperature. Therefore, freezing processes have been developed which take into account the described abnormal thermal behaviour of aqueous samples. But, these processes don't take into account the actual freezing curve of the freezing samples, i.e. they don't use the sample's temperature for the regulatory system. Because of practical problems, the sample's mass is mostly not equal to the mass, the freezing curve has been established for. In this case, the freezing chamber generates a freezing curve of the samples which may be similar to that shown in FIG. 1, resulting in increased damage to the cells. The slightest variations from the freezing curve of the chamber for the particular cell drastically reduces the number of living cells in the sample and produces undesirable ice crystals and other harmful effects. Furthermore, all of the freezing units currently available, freeze laboratory samples only, that is, small samples having a volume no greater than about 2 ml. Consequently, the known freezing systems do not meet the increased requirements of large quantities of cells for broad spectrum therapy.
To be therapeutically effective, large quantities, as for example in the case of medullar cells, lymphocyte cells, granulocyte cells, amounts of about 1.times.10.sup.10 (range 1.times.10.sup.9 to 1.times.10.sup.11) are required and for thrombocytes (platelets) amounts of about 1.times.10.sup.11 (range 1.times.10.sup.9 to 1.times.10.sup.11) are required. These increased requirements can not be met by the prior art, since
(a) the cells must be frozen in volumes of about 100 to 200 ml in each unit sample, as otherwise the loss of time and sterility in filling smaller samples is too great. Steritily is insured in the prior art only, when the techniques of transfusion medicine are used and the storage the small samples is very expensive. For example, the refrigerant costs for storing a vessel of volume of 320 liters are approximately 10,400 DM (about $6,000) annually. The present invention permits the collection and storage of large amounts and at reduced costs.
(b) The therapeutic dose must contain after thawing at least 80% living cells as otherwise a lesser amount of living cells is ineffective in carrying out the vital therapeutic function. In the prior art test cells are sufficient at 50% viability, while generally, the best of the prior art samples do not produce more than 70% viable cells. On the other hand, cells frozen by the present invention are viable concentrations normally between 80-90% and sometimes to about 95% or more. Of course, not only the viable concentrations of cells give therapeutic effectiveness, but also the absolute number of living cells, which is the total recovery. This is the viable concentration times the absolute number of cells ready for transfusion divided by the number of cells originally in the sample before freezing. Though a sample may for example contain more than 70% viable cells, a total recovery may be near 10%. When cells are frozen by the present invention, the total recovery exceeds 80% on an average.
(c) Commercial freezing equipment show temperature fluctuation in the freezing chamber in about a theoretical value of .+-.3.degree. C. This fluctuation in temperature reduces the viability of the cells along the edge zones of the container and reduces the viability even more.
(d) The known commercial freezing units have a temperature/time function which is not influenced by the behavior of the sample itself, but only by the behavior of the freezing chamber. Therefore, the freezing in such equipment will proceed in the desired manner only if the sample volume is not changed relative to the predetermined setting, and irrespective of the actual freezing of the sample itself. Thus, when different sample geometry is used, a new program control must be employed with such freezing units. On the other hand, the control system according to the present invention renders such a step unnecessary.
In order to avoid the foregoing difficulties, and to obtain the objects of the present invention, the present invention proposes to use not only the temperature of the freezing chamber itself but a measured temperature of the sample being frozen for the control of the various phases of the cooling process and to determine both the sample and chamber temperatures simultaneously.